Conservation and Diversity of HMW1 and HMW2 Adhesin Binding Domains among Invasive Nontypeable Haemophilus influenzae Isolates

Abstract

The pathogenesis of nontypeable Haemophilus influenzae (NTHi) begins with adhesion to the rhinopharyngeal mucosa. In almost 80% of NTHi clinical isolates, the HMW proteins are the major adhesins. The prototype HMW1 and HMW2 proteins, identified in NTHi strain 12, exhibit different binding specificities. The two binding domains have been localized in regions of maximal sequence dissimilarity (40% identity, 58% similarity). Two areas within these binding domains have been found essential for full level adhesive activity (designated the core-binding domains). To investigate the conservation and diversity of the HMW1 and HMW2 core-binding domains among isolates, PCR and DNA sequencing were used. First, we separately amplified the hmw1A-like and hmw2A-like structural genes in nine invasive NTHi isolates, discovering two new hmwA alleles, whose sequences are herein reported. Then, the hmw1A-like and hmw2A-like PCR products were used as the template in nested PCR to produce amplicons encompassing the encoding sequences of the two core-binding domains. In-depth sequence analysis was then performed among sequences of each group, with the support of specific computer programs. Overall, extensive sequence diversity among isolates was highlighted. However, similarity plots showed patterns consisting of peaks of relatively high similarity alternating with strongly divergent regions. The phylogenetic tree clearly indicated the HMW1-like and HMW2-like core-binding domain sequences as two clusters. Distinct sets of conserved amino acid motifs were identified within each group of sequences using the MEME/MOTIFSEARCH tool. Since HMW adhesins could represent candidates for future vaccines, identification of specific patterns of conserved motifs in otherwise highly variable regions is of great interest.Nontypeable Haemophilus influenzae (NTHi) is regarded as an important human pathogen, able to cause human respiratory tract disease and, especially in adults, invasive diseases such as meningitis and septicemia (11,12). The initial step in the pathogenesis of NTHi disease involves establishment of bacteria on the rhinopharyngeal respiratory mucosa followed by contiguous spread within the respiratory tract and, occasionally, to sterile sites. A number of adherence factors, including both pilus and nonpilus adhesins, have been identified (24). In almost 80% of NTHi clinical isolates, the HMW proteins are the major adhesins responsible for attachment to human epithelial cells (29). They were originally identified as a group of high-molecular-weight, surface-exposed proteins, predominant targets of human serum antibody response during acute otitis media (1).Given their functional role and their immunogenic character, HMW proteins have gained special consideration as NTHi vaccine candidates. The prototype proteins were detected in NTHi strain 12 and were designated HMW1 and HMW2 (2). They are encoded by separate chromosomal loci, hmw1 and hmw2, respectively, the hmwA gene encoding the structural protein, and two accessory genes, called hm...

Analysis of Genetic Relatedness of Haemophilus influenzae Isolates by Multilocus Sequence Typing

“…Determination of the distribution of some of these islands across the H. influenzae phylogenetic tree is in progress (A. L. Erwin et al, unpublished data) and will add to our understanding of the differences between genetic groups. While in this study we have scored genetic islands simply as present or absent, we recognize that there is substantial sequence heterogeneity for many islands and for several other genes encoding outer membrane proteins (3,6,19,24). Our goal in studying these islands was not to characterize differences between b Phylogenetic groups are included if more than two NTHI strains in the group were examined for the indicated loci.…”

Genome sequencing of disease and carriage isolates of nontypeable Haemophilus influenzae identifies discrete population structure

“…A high level of conservation is also found in the last 740 bases (83% of identity). The overall identity is about 80% (64). The product of hmw1B and hmw2B is 99% identical, and the products of hmw1C and hmw2C show 97% of identity (31).…”

Haemophilus influenzae in children with cystic fibrosis: Antimicrobial susceptibility, molecular epidemiology, distribution of adhesins and biofilm formation

“…Briefly, PCR analysis was carried out by using primers and following PCR conditions as previously described (Giufrè et al, 2006;Cardines et al, 2007). For dot blot analysis, genomic DNA was spotted onto a nylon membrane (Amersham Biosciences, Buckinghamshire, United Kingdom) and hybridized with hmw1A−, hmw2A− and/or hia-specific probes generated by PCR amplifications of the corresponding genes in prototype strains NTHi12 and NTHi 11, respectively (St. Geme et al, 1998).…”